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FIGURE 2: Reactions of Serratia protease-treated platelets. The right hand portion of the figure shows thrombin binding isotherms for control platelets and for platelets treated with lOOng/ml of Serratia protease. Note the absence of high affinity binding and the requirement for an adequate number of binding points to accurately reflect changes in ligand binding. The left hand portion of the figure demonstrates that there is little change in surface expression of GPIba or in vWF/ristocetin reactivity but that ~80% of the Ca2+ response to 0.5nM a-thrombin is lost. Modified from 54, with permission.

-9-8-7-6 UGAND CONCENTRATION (LOG)

FIGURE 2: Reactions of Serratia protease-treated platelets. The right hand portion of the figure shows thrombin binding isotherms for control platelets and for platelets treated with lOOng/ml of Serratia protease. Note the absence of high affinity binding and the requirement for an adequate number of binding points to accurately reflect changes in ligand binding. The left hand portion of the figure demonstrates that there is little change in surface expression of GPIba or in vWF/ristocetin reactivity but that ~80% of the Ca2+ response to 0.5nM a-thrombin is lost. Modified from 54, with permission.

It has been frequently commented upon that the total number of copies of the GPIb-IX-V complex present on platelets (~25,000) vastly exceeds the number of high affinity thrombin binding sites (~50). This apparent discrepancy probably reflects the fact that these high affinity sites are part of this "supercomplex" comprising a dimeric form of the GPIb-IX-V complex and, perhaps, other components. This supercomplex may be a stable structure of all of its individual components or it may reflect an equilibrium binding of monomelic and dimeric forms of the GPIb-IX-V complex.

Recent studies using immobilized glycocalicin, some carried out at 4°C, have provided further confirmation of the role of GPIb as a thrombin receptor but have posed questions regarding the affinity of its interaction with a-thrombin.45,56,57 The reduced temperature used in these studies is of concern since this could affect binding affinity through changes in membrane microviscosity, as discussed above. Moreover, immobilized glycocalicin exhibits only a single thrombin binding site (Kd ~10"8M) whereas binding to purified soluble GPIb-IX and glycocalicin demonstrates both high (Kd ~0.3nM) and moderate affinity (Kd ~50nM) sites.58 Although the moderate affinity receptor is now known to be PARI, these results suggest that immobilization of glycocalicin may prevent the self-association that may be required for expression of a high affinity binding component.

Both the GPIb-IX-V complex and PARI are required to ensure the maximal rate and extent of thrombin-induced platelet activation as measured by the increase in [Ca2+]; and appear to act additively and independently (Fig. 2).27 The thrombin-induced increase in [Ca2+]jWas inhibited by 50% in the presence of antibodies to either GPIb or PARI and was almost completely inhibited by the simultaneous addition of antibodies to both receptors: these results demonstrate that receptors other than GPIb and PARI are unlikely to be involved in thrombin-induced platelet activation. Specifically, the anti GPIb antibodies TM60 and LJib 10 maximally reduced the thrombin-induced increase in [Ca2+]j to 57 ± 13% and 51 ± 4%, respectively, of control values while the anti PARI antibody anti TNA reduced it to 35 ± 13%. In the presence of both TM60 and anti TNA, [Ca2+]j was reduced to 10 ± 5% of the control. This difference from the values obtained with each antibody separately is significant at p<0.05. The fact that Ca2+ mobilization decreases by 50% in the presence of these anti GPIb MoAbs is of importance since, if the role of GPIb were only to facilitate activation through PARI, as has been suggested by several investigators, it would be expected to affect the rate but not the extent of activation as measured by changes in cytoplasmic Cai+ concentration.

Reports that have questioned the relevance of the binding of a-thrombin to the GPIb-IX-V complex in mediating platelet activation have generally reached this conclusion as a result of the use of inappropriate assays. For example, ~22 point binding isotherms are required for the resolution of high, moderate and low affinity thrombin binding sites in a three site model.5 However, 10 point binding isotherms, which would be sufficient for only a one site model, have been the basis for a proposal that GPIb acts as a negative regulator by sequestering small amounts of a-thrombin on the platelet surface.9'10 This model would, in fact, imply that Bemard-Soulier platelets, which cannot sequester thrombin in this manner, should be more sensitive to a-thrombin than control platelets whereas the reverse is known to be the case.

Another study, which concluded that GPIb plays no role in either thrombin binding or thrombin-induced platelet activation, used flow cytometry to measure thrombin binding59 although this technique cannot be used to assay less than about 500 binding sites per cell and is for too insensitive to detect the 50 high affinity thrombin binding sites that occur on platelets.

The cDNA of each of the components of the GPIb-IX-V complex has been cloned and the proteins have been expressed in fibroblasts and other cell types lacking these structures but the mechanism by which the binding of a-thrombin to GPIba initiates platelet activation through the high affinity pathway remains to be determined.

It should be emphasized that, while proteolytically-active thrombin is required for activation through the GPIb-IX-V complex, there is at this time no a priori evidence that a proteolytic event is involved. Conformational changes in solution structure are known to occur in thrombin analogues following derivatization at its active site (PPACK-thrombin) or mutation at the anion binding exosite. These changes include differences in intrinsic fluorescence, interaction with recombinant hirudin peptides and susceptibility to proteolysis.60,61 These changes could abrogate PPACK-thrombin's ability to effect activation although still capable of binding to the receptor.

There are several structural features of GPIb which may play a role in its receptor function and its capacity to induce signal transduction. It was early recognized that glycocalicin, the proteolytic fragment comprising essentially the whole extracellular domain of GPIba, contains binding sites for both a-thrombin and vWF.62 However, the two types of sites have been distinguished by the fact that the high affinity binding form of the receptor can be totally degraded with Serratia marcescens metalloprotease under conditions where vWF responsiveness is essentially unaffected.54 The proximity of the thrombin and vWF binding sites on GPIba may also play a role in the ability of fibrin monomer to facilitate the interaction of GPIba with vWF.63

Further studies45 have localized the thrombin binding domain of GPIba to sequences in the region D269-DM7 which includes a heptapeptide sequence f76 DYYPEE that is also important for the binding of vWF.64 Desulfation or mutation of any one of these three tyrosine residues so that it cannot be sulfated has recently been shown to result in a loss of thrombin binding ability.57,65 These results strongly support a significant role for these sulfation/desulfation processes in initiating thrombin-induced signalling through the GPIb-dependent high affinity pathway.

Hie possible involvement of phosphoiylation/dephosphorylation events at the GPIb-IX-V complex in thrombin-induced platelet activation has not been experimentally established but cannot be unequivocally ruled out. Thrombin-induced platelet activation does not cause measurable changes in the level of phosphorylation at S166 of GPIbp, the only phosphorylated residue in the GPIb-IX complex, but this could reflect the feet that there are only ~50 copies of the high affinity, high molecular weight receptor complex so that changes that might occur in the complexed form of GPIbp could be obscured by the absence of phosphorylation changes in the -30,000 copies of the uncomplexed form.

2.6 Two receptor model

In our earlier model,18 we had inferred from the literature that the high affinity GPIb-dependent receptor was activated by a-thrombin and was coupled to phospholipase A2 while PARI was coupled to phospholipase C and could be activated by the tethered ligand peptide and by both a- and y-thrombins: This hypothesis has been largely confirmed by recent work. The PARI-mediated moderate affinity pathway has been shown to be coupled to both PLC and adenylate cyclase23,67 and this coupling occurs through the G, and G; forms since their activities are stimulated and inhibited, respectively, by TIP.24 Gaq knockout mice fail to aggregate or secrete ATP in response to a-thrombin.68 They do, however, manifest a shape change response indicating that a Gaq-independent mechanism of thrombin-induced activation remains operational presumably through the high affinity pathway. Since TLP activates p38m*pk this kinase may be assigned to the moderate affinity pathway.

The high affinity receptor form of the GPIb-IX complex probably exists as a dimer associated with GPV in the ratio of 2:1 and requires sulfation of specific tyrosine residues in the sequence Y276DYY of GPIba. This receptor mediates the inhibition of thrombin-induced platelet activation by kininogens49 and, in part, thrombin-induced prothrombinase assembly.50 The association of PLA2 with the high affinity pathway mediated by the GPIb-IX-V complex has been supported by the demonstration that the PLA2 zeta isoform (14-3-